Liposomes are completely closed structures comprising lipid bilayer membranes containing an encapsulated aqueous volume. Liposomes may contain many concentric lipid bilayers separated by an aqueous phase (multilamellar vesicles or MLVs), or alternatively, they may comprise a single membrane bilayer (unilamellar vesicles). The lipid bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. In the membrane bilayer, the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer, whereas the hydrophilic (polar) "heads" orient toward the aqueous phase. The basic structure of liposomes may be made by a variety of techniques known in the art.
Liposomes have typically been prepared using the process of Bangham et al., (1965 J. Mol. Biol., 13: 238-252), whereby lipids suspended in organic solvent are evaporated under reduced pressure to a dry film in a reaction vessel. An appropriate amount of aqueous phase is then added to the vessel and the mixture agitated. The mixture is then allowed to stand, essentially undisturbed for a time sufficient for the multilamellar vesicles to form. The aqueous phase entrapped within the liposomes may contain bioactive agents, for example drugs, hormones, proteins, dyes, vitamins, or imaging agents, among others.
Liposomes may be reproducibly prepared using a number of currently available techniques. The types of liposomes which may be produced using a number of these techniques include small unilamellar vesicles (SUVs) [See Papahadjapoulous and Miller, Biochem. Biophys. Acta., 135, p. 624-638 (1967)], reverse-phase evaporation vesicles (REV) [See U.S. Pat. No. 4,235,871 issued Nov. 25, 1980], stable plurilamellar vesicles (SPLV) [See U.S. Pat. No. 4,522,803, issued June 11, 1985], and large unilamellar vesicles produced by an extrusion technique as described in copending U.S. patent application Ser. No. 622,690, filed June 20, 1984, Cullis et.al., entitled "Extrusion Technique for Producing Unilamellar Vesicles", relevant portions of which are incorporated herein by reference.
Liposomes may be used as carriers for a wide variety of materials, for example drugs, cosmetics, diagnostic reagents and bioactive compounds, among others. Liposome compositions to which proteins are conjugated may be designed for both diagnostic and in vivo uses. For example, the ability to produce an antibody-directed vesicle would be a distinct advantage over similar undirected systems (Gregoriadis, G., Trends Pharmacol Sci, 4, p. 304-307, 1983), as would the targetting of a specific receptor or other cell surface feature. Useful applications of these protein-liposome conjugates would be in the selective targeting of cytotoxic compounds entrapped in vesicles to circulating tumor cells (Wolff et.al., Biochim. Biophys. Acta, 802, p. 259-273 1984), or applications of these immunoglobulin-associated vesicles in the development of diagnostic assays. Further applications could result from the targeting of a specific protein-receptor interaction for delivery of active agent to a specific site in a patient. Indeed, protein conjugated liposomes theoretically could be used to target the delivery of any active agent to a site in the patient's system to which the protein will bind. Numerous techniques for the conjugation of proteins to liposomes have already been developed for a variety of purposes including the targeting of drugs via immunoliposomes [See Leserman, et al., Nature. 288, 602 (I980), Heath, et al., Proc. Natl. Acad. Sci. USA, 80, 1377 (1983) and Huang, et al., J. Biol. Chem., 258, 14034 (1983)], diagnostic protocols [See Ishimori, et al., J. Immunol. Methods, 75, 351 (1984) and Ho, et al., J. Immunol.. 134, 4035 (1985)]and liposomal vaccines [See Allison, et al., Nature, 252, 252 (1974)].
Liposomes may be covalently coupled to proteins, antibodies and immunoglobins. Heath et.al. (Biochim. Biophys. Acta., 640, p. 66-81, 1981), describe the covalent attachment of immunoglobulins to liposomes containing glycosphingolipid. Leserman et. al. (Liposome Technology, III, 1984, CRC Press, Inc., CA., p. 29-40; Nature, 288, p. 602-604, 1980) and Martin et. al., (J. Biol. Chem., 257, p. 286-288, 1982) have described procedures whereby thiolated IgG or protein A is covalently attached to lipid vesicles, and thiolated antibodies and Fab' fragments are attached to liposomes, respectively. These protocols and various modifications (Martin et.al, Biochemistry, 20, p. 4229-4238, 1981; and Goundalkar et.al. J Pharm. Pharmacol., 36, p. 465-466, I984) represent the most versatile approaches to coupling. Avidin-coupled and avidin and biotinylcoupled phospholid liposomes containing actinomycin D have successfully targeted tumor cells expressing ganglio-Ntriosylceramide (Urdal et.al., J. Biol. Chem., 255, p. 10509-10516, 1980). Huang et.al. (Biochim. Biophys. Acta.. 716, p. 140-150, 1982) demonstrate the binding of mouse monoclonal antibody to the major histocompatibility antigen H-2 (K), or goat antibody to the major glycoprotein of Molony Leukemia Virus, to palmitic acid. These fatty acid modified IgGs were incorporated into liposomes, and the binding of these liposomes to cells expressing the proper antigens characterized. Other in vitro efforts to specific binding of liposomes coated with specific immunoglobins have been performed (Sharkey et.al., Fed. Proc., 38, 1089, 1979). In still other coupling studies, Rahman et. al. found that tissue uptake of liposomes could be altered by attachment of glycolipids to the liposomes (J. Cell Biol., 83, p. 268a, 1979).
In accordance with a primary use for liposomes, the entrapment of antineoplastic agents inside liposomal bilayers has resulted in more efficacious therapy as compared to direct administration of the drug. (Forssen et.al., Cancer Res., 43, p. 546, 1983; and Gabizon et.al., Cancer Res., 42, p. 4734, 1982). A major problem with the encapsulation of antineoplastic drugs as well as other agents is that many of these drugs have been found to be rapidly released from liposomes after encapsulation. This is an undesirable effect, in view of the fact that toxicity of many of the antineoplastic agents can be significantly reduced through liposome encapsulation as compared to direct administration. See, for example, Forssen et.al. Cancer Res. 43, 546 (1983) and Rahman et.al. Cancer Res , 42, 1817 (1982). In addition, certain pharmacological agents which are favorably delivered in sustained released fashion are not accommodated by standard liposomal delivery systems; many liposomal compositions release the agent too rapidly to provide sustained release delivery.
One answer to the above-described problem is the use of preformed, stable liposomes which maintain the stability and sustained release characteristics of the liposomal system. Liposomal compositions comprising protein-coupled liposomes have produced storage stable liposomes which may be stored stably for an indefinite period, as described in U.S. Patent No. 4,885,172, issued Dec. 5, 1989 entitled "Novel Composition for Targeting, Storing and Loading of Liposomes". These liposomes, which include streptavidin and immunoglobulin coupled to liposomes, may be stored in a dehydrated state, with loading of the liposomes on an "as needed" basis. These protein-coupled liposomes have been loaded with ionizable antineoplastic agents wherein a transmembrane potential is created across the walls of the liposomes and the antineoplastic agent is loaded into the liposomes by means of the transmembrane potential. See, for example, U.S. patent application Ser. No. 749,161, Bally et.al. entitled "Encapsulation of Antineoplastic Agents in Liposomes," filed June 26, -985, relevant portions of which are incorporated herein by reference.
As explained above, protein-liposome conjugates have many potential applications, ranging from diagnostic systems to the targeting of disease states in vivo. As indicated elsewhere [Loughery, et al., Biochim, Biophys Acta., 901, 157 (1987], the coupling of streptavidin to liposomes results in a flexible basic system which subsequently allows the straightforward conjugation of a wide variety of proteins. However, liposome-protein conjugates tend to aggregate during the conjugation process, particularly at high protein to lipid ratios. For example, it has been found that increased amounts of protein [F(ab) fragments]conjugated to liposomes resulted in an increase in the polydispersity of vesicle populations [See Bredehorst R., et al., Biochemistry, 25, 5693 (I986)]. It has also been observed that conditions which increase the coupling efficiency of protein to liposomes, such as increasing the lipid concentration and the ratio of protein to lipid in the coupling incubation step, increase the extent of vesicle-aggregation as observed by negative staining [See Heath, et al., Biochim. Biophys. Acta, 599, 42 (1980)].
Aggregation of protein-liposome conjugates during protein coupling, unfortunately, is a characteristic which impairs the general applicability of this system. This aggregation phenomenon is associated with an increased size of liposomes. It has been observed that the rate of clearance of liposomes from the circulation is dependent on the size of the preparation; the larger the liposome, the faster it is removed from the circulation [See Hunt, A.C., Biochim. Biophys. Acta, 719, 450 (1982) and Sota, et al., Chem. Pharm. Bull., 34, 4244 (1986)]. Because of the tendency of protein liposome conjugates to aggregate, the size of such preparations has tended to be large and thus, circulation times have been somewhat disadvantageous. The clearance of protein-liposome conjugates from the blood has tended to be greater than non-conjugated liposomes of the same size. In addition, aggregated protein-liposome conjugates tend to be poorly taken up by cells via an endocytosis process which may diminish the amount of agent which enters the cells. In diagnostics, the aggregated conjugates tend to precipitate out of solution resulting in potential inaccuracies in diagnosis.
There is, therefore, a need in the art for a general method for producing protein-liposome conjugates of defined size distribution which may be utilized for general targeting applications. Such sized protein-liposome conjugates would be expected to show the favorable characteristics of protein-liposome formulations for targeting active agent delivery, including high cell uptake, or for use in diagnostics, without exhibiting substantial precipitation of aggregated protein-liposome conjugates.
It is an object of the present invention to provide a general method of attaching protein molecules to liposomes to achieve well-characterized sized protein-liposome conjugate systems for general targeting applications.
It is an additional object of the present invention to present a technique for the generation of sized protein-liposome conjugates which should allow ease of conjugating protein to liposome without affecting the binding activity of the protein to which the liposome is conjugated.
It is a further object of the present invention to provide a general method for the generation of protein-liposome conjugates of defined size distribution which can accommodate varying amounts of protein.
It is still a further object of the present invention to provide stable protein-liposome conjugates which are produced by the method of the present invention.
It is still an additional object of the present invention to provide a general method to allow for easy manipulation of the physical size of protein-coupled liposomes without affecting the binding activity of the protein.
It is yet another object of the present invention to enhance the efficiency of the production of sized protein liposome conjugates by providing an efficient coupling technique in combination with stable cross-linkages to increase the in vitro capabilities and stability of the conjugates to more efficiently deliver encapsulated materials to cells.
It is a further object of the present invention to provide sized protein-liposome conjugates which can be stored stably for long periods of time.
It is still another object of the present invention to provide sized protein-liposome conjugates which may be loaded with a bioactive agent using a transmembrane ion potential.